Identification of a domain within the human T-cell leukemia virus type 2 envelope required for syncytium induction and replication

Abstract

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.

FIG. 1

The amino terminus of HTLV-2 p21 is necessary but not sufficient for syncytium formation in BJAB cells. BJAB cells (5 × 10⁵) were transfected with genomic constructs containing chimeric envelopes (shown) as described in Materials and Methods. At day 3 posttransfection, cells were analyzed for syncytia by microscopic analysis. + and − indicate that greater than or less than 10% of the cell population were undergoing cell fusion, respectively. HTLV-2 sequences are depicted by open boxes; HTLV-1 sequences are depicted by dark boxes.

FIG. 2

HTLV-2 replication in BJAB cells requires sequences at the amino terminus of p21. BJAB cells (5 × 10⁵) were transfected with genomic constructs containing chimeric envelopes as described in Materials and Methods. Every 3 days, the medium was changed by allowing the cells to settle and replacing half of the medium. At 3, 7, 14, and 21 days posttransfection, supernatants were collected and cell debris was removed by centrifugation at 3,000 rpm for 5 min as described in Materials and Methods. The amount of secreted HTLV p24 was quantitated by ELISA as described in Materials and Methods. (A) Clones that induced syncytia in BJAB cells (Fig. 1) which included full-length HTLV-2 envelope (II), as well as clones NK, MH, and KB. (B) Clones that did not induce syncytia in BJAB cells (KH, KM, BM, and NKM). (C) Results from the full-length HTLV-2 envelope and the full-length HTLV-1 envelope (NH). The data are representative of three independent experiments. OD₅₅₀, optical density at 550 nm.

FIG. 3

The amino terminus of HTLV-2 p21 is required for syncytium formation in HeLa cells. HeLa cells (10⁶) were infected with wild-type vaccinia virus (WR) or VTF7-3 (MOI = 1.0) for 2 h at 37°C. HeLa cells that were infected with WR were transfected with pEM-ClacZβgAn, a plasmid in which the Escherichia coli lacZ gene has been linked to the T7 promoter. Cells that were infected with VTF7-3 were transfected as described in Materials and Methods with vaccinia virus constructs expressing chimeric HTLV envelopes. At 16 h posttransfection, 10⁵ cells from each of the two sources were mixed and scored for blue syncytia as described in Materials and Methods. These data represent the average of two duplicate wells and are representative of two independent experiments. □, HTLV-2; ▪, HTLV-1.

FIG. 4

The chimeric HTLV envelopes produce equivalent amounts of intracellular gp61. HeLa cells (10⁵) were infected with VTF7-3 (MOI = 1) for 2 h at 37°C and transfected with vaccinia virus constructs expressing chimeric HTLV envelopes as described in Materials and Methods. Cells were harvested 16 h posttransfection. Total cell lysates (10 μg) were separated on SDS–10% polyacrylamide gels and analyzed for the presence of HTLV envelope by Western blot analysis as described in Materials and Methods. Sizes (in kilodaltons) of the molecular weight standards are indicated on the left; gp61 is indicated by the arrow on the right.

FIG. 5

The chimeric HTLV envelopes are expressed efficiently on the cell surface of infected cells. HeLa cells (10⁵) were infected with VTF7-3 for 2 h at 37°C and transfected with vaccinia virus constructs expressing chimeric HTLV envelopes as described in Materials and Methods. Cells were harvested 16 h posttransfection, and 5 × 10³ cells were analyzed by flow cytometry for expression of envelope on the cell surface as described in Materials and Methods. The histograms indicate relative cell number (y axis) as a function of relative amount of gp46 on the cell surface (x axis). Mock-infected cells are represented by the white histogram area, and cells transfected with chimeric HTLV envelopes are represented by the dark histogram area.

FIG. 6

The chimeric HTLV envelopes are similar in the ability to form multimers. HeLa cells (10⁶) were infected with VTF7-3 for 2 h at 37°C and transfected with vaccinia virus constructs expressing chimeric HTLV envelopes as described in Materials and Methods. Cells were harvested 16 h posttransfection and lysed in 500 μl of OGL buffer. (A) Sequence comparison of the amino terminus of HTLV-2 and HTLV-1 p21 proteins. The first 64 aa of HTLV-2 and HTLV-1 p21 are represented. The nonconservative amino acid changes between HTLV-2 and HTLV-1 are boxed. (B) Western analysis of native envelope proteins. Total-cell lysates (20 μl) were separated on a 5 to 20% gradient nondenaturing acrylamide gel with 0.01% SDS in the running buffer as described in Materials and Methods. HTLV envelope was detected by Western blot analysis as described in Materials and Methods. Sizes (in kilodaltons) of the molecular weight standards are indicated on the left; the multimeric forms of HTLV envelope are indicated by the arrow on the right. (C) Western analysis of denatured envelope proteins. Total-cell lysates (20 μl) were heated to 100°C for 5 min and separated on a 5 to 20% gradient nondenaturing acrylamide gel with 0.01% SDS in the running buffer as described in Materials and Methods. HTLV envelope was detected by Western blot analysis as described in Materials and Methods. Sizes of the molecular weight standards are indicated on the left; gp61 is indicated by the arrow on the right.